The principle of immunohistochemistry technology: antigen-antibody reaction, that is, the principle of specific binding of antigen to antibody, the color of the labeled antibody (fluorescein, enzyme, metal ion, isotope) is determined by chemical reaction to determine the antigen in tissue cells. (polypeptides and proteins) for their localization, qualitative and quantitative studies.
It is well known that the binding between an antibody and an antigen is highly specific. Immunohistochemistry is the use of this feature, that is, first extract some of the chemical substances in tissues or cells , use them as antigens or haptens to deimmunize experimental animals, prepare specific antibodies, and then use this antibody (first The antibody is used as an antigen to immunize the animal to prepare a second antibody, and is treated with an enzyme (usually horseradish peroxidase) or biotin, and then combined with the aforementioned antigen component to form an antigen-? primary antibody-second antibody complex. The antigen is amplified, and since the immune complex formed by binding the antibody to the antigen is colorless, the antigen-antibody reaction site must also be displayed by means of histochemical methods (the usual developer DAB is shown as brown-yellow particles). Through antigen-antibody reaction and color reaction, the chemical components in cells or tissues are displayed, and the antigen-antibody reaction products occurring in the cells can be clearly seen under the microscope, so that the distribution and content of certain chemical components can be determined in situ in the cells or tissues. . Any substance or antigen that can be used as an antigen or hapten in a tissue or cell, such as a protein, a polypeptide, an amino acid, a polysaccharide, a phospholipid, a receptor, an enzyme, a hormone, a nucleic acid, and a pathogen, can be detected by a corresponding specific antibody.
Immunohistochemistry technology experiment classification (common)
1. Immunofluorescence method
The earliest established immunohistochemical technique. It uses the principle of antigen-antibody specific binding, first labeled known antibodies with fluorescein, as a probe to examine the corresponding antigen in cells or tissues, and observed under a fluorescence microscope. When the fluorescein in the antigen-antibody complex is irradiated with the excitation light, a certain wavelength of fluorescence is emitted, thereby determining the localization of an antigen in the tissue, and further quantitative analysis. Because immunofluorescence technology is highly specific, sensitive, fast and simple, it is widely used in clinical pathology diagnosis and testing.
2, immune enzyme labeling method
The immunoenzymatic labeling method is a technique developed in the 1960s following immunofluorescence. The basic principle is that the enzyme-labeled antibody acts on the tissue or cells, and then the substrate of the enzyme is added to form a colored insoluble product or a particle having a certain electron density, and the surface of the cell and various cells are irradiated by light microscopy or electron microscopy. The antigen component was subjected to localization studies. The immunolabeling technology is currently accurate in positioning, good in contrast, and long-term preservation of stained specimens, suitable for light and electron microscopy studies. The development of immunolabeling methods has been very rapid, and a variety of labeling methods have been derived. Currently, PAP (peroxidase-antiperoxidase) and ABC (albumin) are widely used in pathological diagnosis. Biotin-peroxidase complex), SP method (streptavidin-peroxidase), ready-to-use two-step method (polymer link), and the like.
3. Immunocolloidal gold technology
The immunocolloidal gold technique uses a special metal particle such as colloidal gold as a marker. Colloidal gold refers to a gold hydrosol that adsorbs proteins quickly and steadily without a significant effect on the biological activity of the protein. Therefore, using a colloidal gold-labeled primary antibody, secondary antibody, or other molecule that specifically binds to an immunoglobulin (such as Staphylococcal Protein A) can be used as a probe to characterize, localize, or even quantify antigens in tissues or cells. the study. Because colloidal gold has particles of different sizes and the electron density of colloidal gold is high, the immunocolloidal gold technique is particularly suitable for single-label or multi-label localization studies of immunoelectron microscopy. Since colloidal gold itself is light to deep red, it is also suitable for light microscopy. For example, the silver-enhanced immunogold and silver rule is more convenient for light microscopy.
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